Evaluating therapeutic potential of NR2E3 doses in the rd7 mouse model of retinal degeneration

Retinitis Pigmentosa is a leading cause of severe vision loss. Retinitis Pigmentosa can present with a broad range of phenotypes impacted by disease age of onset, severity, and progression. This variation is influenced both by different gene mutations as well as unique variants within the same gene. Mutations in the nuclear hormone receptor 2 family e, member 3 are associated with several forms of retinal degeneration, including Retinitis Pigmentosa. In our previous studies we demonstrated that subretinal administration of one Nr2e3 dose attenuated retinal degeneration in rd7 mice for at least 3 months. Here we expand the studies to evaluate the efficacy and longitudinal impact of the NR2E3 therapeutic by examining three different doses administered at early or intermediate stages of retinal degeneration in the rd7 mice. Our study revealed retinal morphology was significantly improved 6 months post for all doses in the early-stage treatment groups and for the low and mid doses in the intermediate stage treatment groups. Similarly, photoreceptor function was significantly improved in the early stage for all doses and intermediate stage low and mid dose groups 6 months post treatment. This study demonstrated efficacy in multiple doses of NR2E3 therapy.

The long-term effects of NR2E3 on the clinical phenotype of rd7 animals were evaluated following subretinal delivery of AAV5 during early or intermediate stages of rd7 clinical disease progression.Animals were treated with either a low, mid, or high dose of AAV5-hNR2E3 at P30 or 3 months of age, and evaluated 1-, 3-, and 6-months post treatment to monitor for any longitudinal adverse effects of NR2E3 on the rd7 animals.The rd7 fundus phenotype has pan retinal spots that lack a consistent spatial or numerical pattern; with variability in the density of spots.These spots fade as degeneration progresses; thus, while it is not useful to quantify the number of retinal spots, the fundus can be used to qualitatively evaluate potential signs of gross abnormalities or inflammation due to treatment, such as retinal scarring, by comparing treated eyes with uninjected and mock injected eyes.Early-stage fundus analysis of rd7 animals treated with NR2E3 at P30 revealed no long-term negative effects such as an increase in pan retinal spots or inflammation in the fundus phenotype for all doses (Fig. 1).Similarly, longitudinal monitoring of intermediate stage treatment (rd7 animals treated at 3-months of age) with NR2E3 revealed no gross abnormalities in the fundus phenotype for all three therapeutic doses (Fig. 2).OCT was performed to track in life observation of any potential adverse effects.No adverse effects were observed in 1-, 3-, and 6-month post early stage (Fig. 1B) and intermediate stage (Fig. 2B) NR2E3 treated rd7 mice.Microglial cells are the main constituents of the immune cell population in the retina and the main initiators of the inflammatory response in the retina [51][52][53][54] .Our prior studies showed that rd7 mice do not undergo inflammation 17 .In order to determine if AAV5-hNR2E3 treatment will induce an inflammatory response through microglial activation, we examined the expression of IBA1, a marker of microglial activation.The results show that there is no evidence of microglial activation reflecting absence of inflammation in NR2E3-treated rd7 mice, similar to normal B6 and untreated rd7 mice (Supplemental Fig. S1).

AAV delivery of NR2E3 in different doses in rd7 animals during early or intermediate disease progression restores retinal morphology
Histology analysis of rd7 animals dosed with AAV5-hNR2E3 during early or intermediate disease progression showed improved retinal morphology.rd7 mice exhibit a slow, progressive, concomitant degeneration of rod and cone photoreceptors, leading to resolution of the whorls over time.A normal mouse retina consists of 10-12 layers of cone and rod photoreceptor nuclei making up the ONL and 5-6 layers of inner retinal cells making up the inner nuclear layer (INL) 16 .In the rd7 model, abnormal morphology presents with whorls and rosettes in the ONL caused by over-proliferation of blue opsin expressing cone cells followed by slow, progressive photoreceptor degeneration 18,50 .The whorls and rosettes begin to resolve and flatten by 5 months of age as degeneration progresses 50 .The non-whorl regions of the retina are the most important for tracking degeneration and rescue as abnormal blue cone proliferation has not disrupted the normal thickness of this region prior to disease onset.Hematoxylin/Eosin (H/E) staining showed fewer whorls and rosettes and increased ONL thickness in P30 treated retinas for all doses over 6 months compared to untreated retinas (Fig. 3, lower magnification in Supplemental Fig. S2).H/E analysis of rd7 animals treated with NR2E3 at 3-months of age similarly demonstrated a reduction in whorls and rosettes and increased ONL thickness in treated retinas for all doses compared to untreated maintained over 6 months (Fig. 4).Some whorls and rosettes will still be present in treated retinas if they were present prior to NR2E3 administration halting proliferation and degeneration.Additionally, to discern the impact of rescue, the ONL in the non-whorl and rosette regions of rd7 animals treated at P30 or 3-months of age and collected 6-months after treatment were counted and compared with untreated retinas.Statistically significant rescue of photoreceptor cells in the non-whorl regions was observed compared to untreated animals in both early and intermediate administration groups except for the intermediate stage high dose treatment (Fig. 5; p < 0.05 to 0.0001).Both the P30 and 3-month injected low dose groups rescued similar amounts of photoreceptors.Interestingly, animals treated at P30 showed rescue to normal ONL thickness (around 10-12 layers) for all doses, compared to mid and high dose animals treated at 3-months of age that showed a more modest rescue of around 8-9 layers.This is likely due to degeneration already occurring when treatment was administered at the intermediate progression phase (3-months of age) and thus fewer cells remained that could be rescued.These data suggest that NR2E3 is effective at halting degeneration when administered at early or a later stage of disease for all doses except the high dose later stage, and for at minimum 6 months after treatment.

AAV5-hNR2E3 improved scotopic ERG responses in rd7 animals treated during early or intermediate progression
The rd7 mice exhibit progressive loss of rod and cone function as measured by abnormal ERG responses with significant loss of function between 6-12 months of age 18,50,55 .Our previous studies of rd7 mice treated with Nr2e3 showed improved ERG responses measured up to 3-months post treatment 16,18 .In this study, dark-adapted and light-adapted ERGs were used to assess photoreceptor function of treated rd7 retinas by examining rodand cone-driven responses up to 6-months post treatment (Fig. 8A-D; Supplemental Fig. S3).Similarly to cell counts, rd7 animals treated during early stage (P30) progression demonstrated statistically significant rescue of dark-adapted photoreceptor function in low, mid and high dose treated retinas (p < 0.0001 for all doses) compared to untreated 6 months post treatment (Fig. 8A).rd7 mice treated at intermediate stage (3-months of age) of disease only showed significant rescue 6-months post treatment for the low and mid dose groups compared to untreated retinas (Fig. 8A, p < 0.0001).A significant improvement in the dark-adapted a-wave function and light-adapted b-wave function was also observed in early and intermediate mid dose treated rd7 retinas compared with untreated retinas (Fig. 8B,C).Significant improvement in photoreceptor function was often not detectable until 6-months post treatment for both administration time points; this could be attributed to the delay in expression of AAV5-hNR2E3 42,56 .The appearance of improved function and lack of significance for some of the doses and time points could be affected by technical aspects of delivery and measuring function via ERG.The focal delivery of NR2E3 via subretinal injection will only rescue a localized portion of the retina whereas full-field ERG measures overall photoreceptor function across the entire retina.The different levels of rescue observed in ERGs for each of the doses and time points is the result of the varying amounts of rescue achieved by localized delivery.Interestingly, animals injected at P30 showed increased improvement in ERG response over time for all doses, perhaps supporting the idea that earlier therapeutic intervention has a chance of improving clinical outcome.

Discussion
The outcomes of inherited diseases such as RP are subject to the influence of several factors including allelic heterogeneity, environment, epigenetic factors, and gene mutations [57][58][59][60] .Nr2e3 is a powerful nuclear hormone receptor that modulates several key gene networks important for maintaining homeostasis in the retina including apoptosis, cell survival, ER stress, immunity, metabolism, neuroprotection, oxidative stress, and phototransduction 16,61 .Our recent study demonstrated that administering Nr2e3 gene therapy prior to disease onset or during early disease rescues degeneration in the rd7 mouse model which has an Nr2e3-associated gene mutation 16 .This current study was a pharmacological preclinical study that informs dose selection and longitudinal effectiveness of NR2E3 therapeutic.The novel focus of this study was to examine the efficacy and longitudinal effects of dosage and early versus intermediate therapeutic intervention.All doses showed improvement of photoreceptor survival, and retinal function as well as partial restoration of retinal structure.Furthermore, no gross abnormalities were observed with any of the doses.Sustained improvement was observed up to 6 months after treatment in early and intermediate disease stage groups.It is important to note that focal delivery of NR2E3 via sub-retinal injection does not rescue the entire retina and manifests as regions of rescue localized around the injection site.This is demonstrated in the functional, histological, and molecular analysis.Localized rescue of photoreceptors illustrate why statistically significant improvements in retinal morphology were observed compared to more limited significance in full field ERGs that measures function across the entire retina.ERGs are also very technically sensitive, and variation demonstrated by the error bars is likely a result of different personnel performing the experiments.Additionally, that it can take AAVs up to 2-4 weeks to begin showing expression likely contributes to the observations of statistically significant improvements after 6 months irrespective of disease stage at time of administration 42,56 .
The rd7 mouse is a functional null representing varied phenotypes of recessive NR2E3-associated retinal disease that encompasses both RP and ESCS 18,19,23,26,50 .The rd7 mouse is unique in that it has two distinct phenotypes: a developmental over-proliferation of blue cones followed by a slow progressive degeneration of all photoreceptors.It is important to note, rd7 mice, unlike many other RP models, actually start with relatively normal expression of rhodopsin that degenerate over time along with cones.It has been suggested that rd7 mice possess some hybrid photoreceptor cells that co-express blue opsin and rhodopsin 62,63 ; however, our prior studies could not confirm these findings nor did we observe any similarities between our individually labeled IHCs from this study (Supplemental Fig. S4) 17,64 .Regardless, rd7 mice have a developmental problem of over-proliferation of blue opsin expressing cone cells due to lack of Nr2e3 in retinal progenitors 17 .Once matured, rd7 photoreceptor cells degenerate, and that is likely due to disruption of Nr2e3 function in many pathways associated with normal function of rod and cone photoreceptor cells.The mechanism of disease in rd7 thus involves the lack of functional Nr2e3 protein that disrupts progenitor cell proliferation, rod and cone differentiation, and rod and cone function and survival 17,18 .
Our work and the work of others demonstrates that NR2E3 plays a role in both the developing and adult retina as well as functions as either an activator or repressor in different photoreceptor development stages 17,64,65 .Nr2e3 targets genes involved in key homeostasis pathways like cell survival, apoptosis, phototransduction, immunity, neuroprotection, ER stress, oxidative stress, metabolism, and immunity which could be a contributor to the degeneration of rods and cones following blue cone over-proliferation in rd7 mice when Nr2e3 proteins are nonfunctional 16 .Importantly, our recent studies show that many RP models have little to no expression of key retinal transcription factors (including Nr2e3.CRX, Nrl, Rora, Nr1d1) and are reset by NR2E3 therapy 16 .Taking the rd7 disease phenotypes into consideration, and as we treated post photoreceptor development (P30 or 3 months).NR2E3, like all NHRs, regulates multiple genes in pathways impacting cell homeostasis [66][67][68] , as demonstrated in our prior study 16 .In the current study, the mechanism of NR2E3 rescue of retinal degeneration in rd7 is likely occurring similarly through modulation of the Nr2e3 regulated genes and gene networks to a more homeostatic state that attenuates degeneration 16,17,23,64 .
As expected, earlier treatment of rd7 mice with NR2E3 is more effective at alleviating or ameliorating disease progression.Treatment during early disease state showed better sustained rescue over time and was able to return ONL thickness and ERG responses to normal levels.It is imperative to note that NR2E3 is a promising treatment for early or intermediate stage degeneration meaning that it can be used to treat early-stage diagnosis patients and patients that have already been diagnosed and progressed to the later stages of degeneration, which is uncommon for many therapeutics.NR2E3 was also able to provide rescue for at least 6 months in both progression stage treatments and rescued photoreceptor survival and function to near normal levels in the intermediate stage.
In conclusion, this study demonstrates that AAV5-hNR2E3 in various doses can treat early and intermediate stage retinal degeneration in the rd7 mouse for at least 6 months.The results reveal that NR2E3 gene therapy in the retina can halt degeneration and improve photoreceptor survival and function.Future studies will evaluate whether NR2E3 reverses degeneration and efficacy of combination therapies.Further studies will also investigate the effectiveness of NR2E3 therapeutic in other non-NR2E3-associated retinal diseases.The results of this pharmacology study are translational to dosage selection of NR2E3 as a therapeutic agent, longitudinal therapeutic effectiveness, and treatment effectiveness in early and intermediate stage NR2E3-associated retinal disease in clinical trials.Following this study, clinical trials of AAV5-hNR2E3 were initiated and are currently underway to examine the safety and efficacy of the therapeutic for treating various forms of Retinitis Pigmentosa and Leber Congenital Amaurosis (ClinicalTrials.govIdentifier: NCT05203939).

Animal maintenance
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, as well as the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.All authors complied with the guidelines for Animal Research: Reporting of In Vivo Experiments (ARRIVE).Animals were housed and bred under standard conditions, temperatures within 68-74°F and 12-h light/12-h dark cycles, in the Schepens Eye Research Institute vivarium for the duration of this study.The Schepens Eye Research Institute Animal Care and Use Committee (Protocol Number: 2020N000178) approved animal use and procedures for this study in compliance with the Animal Welfare Act Regulations.C57BL6/J (B6; Jax stock #000664), and Nr2e3 rd7 /J (rd7; Jax stock #002139) mice were ordered from Jackson Laboratories, Bar Harbor, ME.   www.nature.com/scientificreports/

Scientific rigor and reproducibility
G*Power software analysis was used to conduct a power calculation for estimating the required sample size for each analysis described (G*Power, version 3.1, https:// www.psych ologie.hhu.de/ arbei tsgru ppen/ allge meine-psych ologie-und-arbei tspsy cholo gie/ gpower).Based on means and standard deviation previously defined in published studies, a minimum of 4 animals were used per experimental group to provide 90% power and 30% difference at a significance level of 0.05.Every procedure was performed using a standardized protocol.This study was performed by several trained individuals in a double blinded and randomized manner to prevent any bias.At least 4-5 biological replicates were studied for each dose and time point to achieve statistical significance.Animals with cataracts, unresolved surgical trauma, or that died prematurely were excluded from the study.Based on these criteria, approximately 20% of the 120 treated animals in this study were excluded from the final analysis.Males (~ 47.37%) and females (~ 52.63%) were used equally, and a gender bias was not observed.

Statistical analysis
Graphs were analyzed for statistical significance using a two way analysis of variance (ANOVA) (GraphPad Prism, version 9.0, https:// www.graph pad.com/).Comparisons were made between the mean peak scotopic or photopic a-or b-wave amplitudes of untreated and treated animals for each treatment and collection time point.Analysis of the differences between the 6-month post time point histological and opsin-positive cell counts, www.nature.com/scientificreports/and mean fluorescence intensity of opsins was also performed for each dose compared to the untreated group (Supplemental Tables S1-S10).

Genotyping
Mouse tail biopsy samples were used for DNA isolation using a quick lysis sodium hydroxide method.Isolated DNA samples were amplified using the primers nr4F: GTA GCC TCT CCT GCT CTG GCAG and rd7del4R: CAG GTT GGA AAA CAC AGG CAAG 18 .For PCR amplification approximately 35 ng of DNA was used in a 10 μl reaction volume containing 10× buffer with MgCl 2 , 40 mM dNTP mix, 10 μM each of forward and reverse primer, and 5 U/ml AmpliTaq DNA polymerase.Reactions were denatured at 95 °C for 3 min followed by 30 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 42 s and a final extension at 72 °C for 5 min.Amplicons were separated using a 2% agarose gel and visualized under UV light after staining with ethidium bromide.The wild-type amplicon is 339 base pairs (bp) in size and the mutant amplicon is 239 bp.As the rd7 mutation is a 9 kb line element insertion in the region amplified, it will not produce an Nr2e3 PCR amplicon under these conditions, and the amplicon from rd7 mice is a pseudogene 18,49 .

AAV5-hNR2E3 dose preparation
The drug product consists of adeno associated virus 5

Subretinal injection
AAV5-hNR2E3 was delivered by subretinal injection into the right eye of rd7 mice.Mice were injected at either P30 to treat early-stage disease progression or 3-months of age to treat intermediate progression.Control injections consisted of no injection in the contralateral (left) eye, and mock injection in the contralateral eye with saline buffer (Supplemental Fig. S5).Adults aged to P30 or 3 months old were anesthetized by intraperitoneal (IP) injection using a ketamine/xylazine mixture.A sterile 30G needle was used to make an incision in the sclera just posterior to the limbus, and a Hamilton syringe with a blunt 33G cannula attached was inserted into the incision 16 .A total volume of 0.5 µl of therapeutic product was manually injected into the subretinal space of the adult mice for each dosage group: low (1 × 10 8 vg/eye), medium (1 × 10 9 vg/eye), and high (4 × 10 9 vg/eye) dose.
Accessing the central retinal region with an injection in the mouse is difficult.Therefore, the location of rescue often appeared between the peripheral and central retina, sometimes extended into the central retina based on the degree of rescue achieved by the focal injection.

Clinical examination
All animals (treated, untreated, wildtype (WT) B6 controls) underwent fundus examination and optical coherence tomography (OCT).Animals were anesthetized with a ketamine/xylazine mixture by IP injection and 1% tropicamide was used to dilate pupils.The Micron III Retinal Imaging Camera (Phoenix Research Laboratories, Pleasanton, CA, USA) and Stream Pix software (version 5, https:// www.norpix.com/ produ cts/ strea mpix/ strea mpix.php) were used for fundus imaging.The Bioptigen OCT scanner (Bioptigen Inc, Durham, North Carolina, USA) and software (Envisu Invivovue, version 2, https:// www.leica-micro syste ms.com/ produ cts/ surgi cal-micro scopes/ p/ envisu-r-class/) were used to perform OCT.A mounting tube with a bite bar restrained mice while the real time OCT image was used for alignment of the retina.Each eye had four rotational cross section scans (nasal-caudal and dorsal-ventral) taken with 100 series/scan 18 .The Bioptigen OCT software analyzed the retinal scans and representative images were taken of the central retina near the optic nerve.

Electroretinography
Mice were examined through electroretinography analysis as previously described 16,17 .Animals were darkadapted overnight or for a minimum of 4 h before being anesthetized.Anesthesia was administered through an IP injection of a mixture of ketamine and xylazine, and pupils were dilated with 1% tropicamide.Genteal was used as a lubricant to prevent cataracts forming during the procedure.The reference electrode was placed subcutaneously in the forehead and the ground electrode was inserted subcutaneously in the tail base.Gold loop electrodes (Diagnosys LLC) were placed on the corneal apex.The Espion Visual Electrophysiology System (Diagnosys LLC, Lowell, MA, USA) performed dark-and light-adapted ERGs.Dark-and light-adapted responses were recorded according to the same protocol used previously 16 .Briefly, responses were recorded at intensities between 0.000249 and 24.1 cd s/m 2 in 4.0 log intensity increments for dark-adapted.Using the same 4.0 log intensity difference, light-adapted responses were obtained between 0.1 and 25.6 cd s/m 2 following 7 min of adaptation to background light.The peak a-and b-wave amplitude from each treated animal was selected between intensities of 0.061 cd s/m 2 and 24.1 cd s/m 2 for scotopic responses and 0.1 cd s/m 2 and 25.6 cd s/m 2 for photopic responses, averaged for each dose and time point, and plotted in comparison to peak amplitudes from wild type and untreated rd7 mice.

Histology
Histological analysis was performed as previously described 16 .Directly following euthanasia, a cautery mark was made on the eyes for dorsal orientation and then eyes were enucleated.

Figure 5 .
Figure 5. AAV5-hNR2E3 treatment reduces ONL degeneration from disease progression in rd7 animals at all doses.Outer nuclear layer (ONL) thickness of animals injected at P30 (early) or 3-months of age (intermediate) and collected 6-months post injection (animal ages 7 months and 9 months) were compared with the non-whorl region of untreated rd7 animals of the same age.Early AAV5-hNR2E3-treated rd7 animals untreated vs low dose p < 0.006, untreated vs mid dose p < 0.0001, untreated vs high dose p < 0.009.Intermediate AAV5-hNR2E3treated rd7 animals untreated vs low dose p < 0.0001, untreated vs mid dose p < 0.04; low dose vs mid dose p < 0.02; low dose vs high dose p < 0.002.Dots represent individual data points for each experimental group.Serial section of central retina counted over 100 µm.Low Dose = 1 × 10 8 v/gc; Mid Dose = 1 × 10 9 v/gc, High Dose = 4 × 10 9 v/gc.Results are mean ± SEM, n ≥ 5.

Figure 8 .
Figure 8. AAV5-hNR2E3 improved peak scotopic b-wave amplitude in rd7 mice for all doses.C57BL6/J (B6) animals were used for wild-type controls.(A) The scotopic b-wave amplitudes of rd7 animals injected at P30 were assessed 1-, 3-, and 6-months post injection for the low, mid, and high dose (animal ages 2, 4, and 7 months).rd7 animals injected at 3-months of age were similarly assessed 1-, 3-, and 6-months post injection for the low, mid, and high dose (animal ages 4, 6, and 9 months).Treated eyes showed significant improvement of retinal function at the 6-month post-injection time points for both the 1 month-injected low and mid dose groups and 3 month-injected mid dose group when compared with age-matched untreated eyes.Intermediate AAV5-hNR2E3 treated 3 months post rd7 mice untreated vs mid dose p < 0.05.Early AAV5-hNR2E3 treated 6 months post rd7 mice untreated vs low dose p < 0.003, untreated vs mid dose p < 0.0001 and untreated vs high dose p < 0.02.Intermediate AAV5-hNR2E3 treated 6 months post rd7 mice untreated vs low dose p < 0.03 and untreated vs mid dose p < 0.0001; low vs mid dose p < 0.04, and mid vs high dose p < 0.0002.Dots represent individual data points for each experimental group.(B) The scotopic a-wave amplitudes of rd7 mice receiving early or intermediate NR2E3 treatment.Early NR2E3 treated 6M post rd7 mice untreated vs mid dose p < 0.009.Intermediate NR2E3 treated 6M post rd7 mice untreated vs mid dose p < 0.003 and mid dose vs high dose p < 0.02.(C) The photopic b-wave amplitudes of early and intermediate NR2E3-treated rd7 mice assessed at 1, 3 and 6 months post-treatment.Early NR2E3 treated 6M post rd7 mice untreated vs mid dose p < 0.002.Intermediate NR2E3 treated 6M post rd7 mice untreated vs mid dose p < 0.007 and mid dose vs high dose p < 0.005.(D) The photopic a-wave amplitude of early and intermediate NR2E3 treated rd7 mice at 1, 3 and 6 months post-treatment.Low Dose = 1 × 10 8 v/gc; Mid Dose = 1 × 10 9 v/gc, High Dose = 4 × 10 9 v/gc.Results are mean ± SEM. n ≥ 5.